The neurotransmitter serotonin (5-hydroxytryptamine or 5HT) plays an important role in numerous behavioral and physiological functions including mood, sleep, appetite, memory, vasoconstriction and addiction. Presynaptic serotonin transporters (SERTs) are responsible for efficient clearance of 5HT from synaptic spaces and are the primary molecules involved in terminating 5HT synaptic signaling. SERTs are high-affinity molecular targets for multiple antidepressants, and drugs of abuse including cocaine, amphetamine and MDMA "ecstasy." Altered SERT function and SERT gene polymorphisms have been implicated in anxiety, depression, suicide, autism and substance abuse. Despite decades of exogenous manipulation of SERTs for clinical ends, we have little knowledge of the endogenous mechanisms that regulate SERT activity. Recently, I have begun to identify mechanisms by which SERTs are regulated endogenously. I found that SERT activity is acutely regulated by protein kinase C (PKC) and protein phosphatase 2A (PP2A) linked pathways, that this regulation occurs primarily through membrane trafficking, and is accompanied by rapid transporter phosphorylation. Moreover, PKC-dependent regulation is impacted by the transporter's own intrinsic activity and is modulated by 5HT, antidepressants, cocaine and amphetamines. The focus of this proposal is to characterize thoroughly the SERT phosphorylation mediated by PKC and Ca2+/calmodulin-dependent kinase II (CaMKII) in relation to functional transporter regulation by combining molecular biologic and biochemical approaches. The specific aims to achieve these goals are: 1) to characterize the PKC- and CaMKII- mediated SERT phosphorylation, and to identify presynaptic receptors and pharmacological stimuli that regulate SERT function and 2) to characterize the role SERT phosphorylation mediated by PKC and CaMKII and dephosphorylation in SERT function, in terms of (a) reversibility and specificity of SERT phosphorylation and dephosphorylation in relationship to SERT activity and sequestration, (b) the domains and sites of SERT phosphorylation in relationship to regulated sequestration and 5HT translocation, and (c) the interaction of SERT substrates and antagonists in the regulation of SERT phosphorylation and expression. These studies will provide critical insights to the role(s) of PKC and CaMKII, and phosphatases in SERT functional regulation and may provide new mechanisms relevant for the understanding of drug action and altered regulation that contribute to mental illness.